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Dendritic cells (DCs) loaded with tumor-associated antigens (TAAs) are known to be crucial for the antitumor response and are still included in various treatment regimens in cancer immunotherapy research. In the present study, a cell-based protocol was evaluated, involving the use of original DNA constructs encoding the wide range of TAA epitopes expressed on different epithelial cancers. The constructs were transfected into in vitro-generated DCs of patients with various types of cancer, including breast, colorectal and non-small cell lung cancer. The direct cytotoxicity assay of effector cells, activated with the transfected DCs, revealed a significant increase in cytotoxicity against autologous tumor cells. The use of DNA constructs encoding a large number of TAAs for insertion into DCs in vitro, aiming to activate a T-cell response may prove to be a reliable and unified approach for immunotherapy and for the prevention of relapse in patients with epithelial cancers.

Background. Nonspecific immunosuppressive therapy for graft rejection and graft-versus-host disease (GVHD) is often accompanied by severe side effects such as opportunistic infections and cancers. Several approaches have been developed to suppress transplantation reactions using tolerogenic cells, including induction of FoxP3+ Tregs with antigen-loaded dendritic cells (DCs) and induction of CD4+IL-10+ cells with interleukin IL-10-producing DCs. Here, we assessed the effectiveness of both approaches in the suppression of graft rejection and GVHD. Methods. IL-10-producing DCs were generated by the transfection of DCs with DNA constructs encoding mouse IL-10. Antigen-loaded DCs from C57BL/6 mice were generated by transfection with DNA constructs encoding antigenic determinants from the H2 locus of CBA mice which differ from the homologous antigenic determinants of C57BL/6 mice. Results. We found that both IL-10-producing DCs and antigen-loaded immature DCs could suppress graft rejection and GVHD but through distinct nonspecific and antigen-specific mechanisms, respectively. Discussion. We provide data that the novel approach for DCs antigen loading using DNA constructs encoding distinct homologous determinants derived from major histocompatibility complex genes is effective in antigen-specific suppression of transplantation reactions. Such an approach eliminates the necessity of donor material use and may be useful in immunosuppressive therapy side effects prevention.

Melanoma is a tumor that forms as a result of malignant transformation of melanin-producing pigment cells (melanocytes). It is the most aggressive form of skin cancer and is characterized by high resistance to chemotherapeutic drugs, which results in a need to explore alternative methods for this disease therapy. Currently, new approaches to cancer treatment are being intensely developed, oncolytic immunotherapy being one of them. This approach consists in using viruses as targeted tumor-specific cytolytic agents capable of stimulating both tumor-specific and nonspecific immune responses. A considerable body of research is currently aimed on improving the immunostimulatory properties of viruses by inserting the genes encoding immunomodulatory proteins or tumor-specific antigenic determinants into viral genomes. For melanoma, the highest number of tumor-associated antigens (TAAs) has been identified, which serve as the basis for the development of anti-tumor DNA vaccines. The immunogenicity and efficacy of these drugs, however, remain low. The bottlenecks in using DNA vaccines to treat cancer are considered to be imperfect design of polyepitope constructs, as well as inefficient delivery of therapeutic molecules directly to the target cells. A partial solution to these problems may be represented by the use of oncolytic viruses as vectors for the delivery of artificial immunogens. The recombinant vaccinia virus was obtained by transient dominant selection. The cytolytic activity of the obtained virus was tested using the MTT assay. The oncolytic activity of the virus was assessed in the mouse xenograft model obtained using malignant SK-Mel-28 cells. This paper reports the production of a recombinant L-IVP_oncoM virus, the oncolytic virus for the delivery of anticancer therapeutic genes into the cells, on the basis of the vaccinia virus strain L-IVP. Toward this end, the gene encoding the Granulocyte-macrophage colony-stimulating factor (GM-CSF) and the artificial gene encoding a polyepitope immunogen containing the epitopes of the antigens over-expressed in melanoma cells were inserted into the virus genome. These insertions were located in the proximity of the genes encoding thymidine kinase (J2R) and viral growth factor (C11L), respectively. The properties of L-IVP_oncoM were studied in in vitro experiments using cell cultures of various origin and in the in vivo experiments using the mouse xenograft model. The basic experiments to assess the biological properties of the obtained L-IVP_oncoM, which are necessary to characterize the oncolytic virus, have been carried out.

Dendritic cells (DCs) loaded with tumor-associated antigens (TAA) are known to be the important agents in antitumor response realization and still figure in lots of treatment schemes in cancer immunotherapy research. Here, we evaluated a cell-based protocol involving the use of original DNA constructs encoding the wide range of TAA epitopes expressed on different epithelial cancers. The constructs were transfected into ex-vivo-generated DCs of cancer patients (breast cancer, colorectal cancer, and non-small cell lung cancer). Direct cytotoxicity assay of effector cells, activated with the transfected DCs, showed a significant increase in the cytotoxicity against autologous tumor cells. The use of DNA-constructs encoding a large number of TAA’s for the in vitro DC loading to activate the T cell response could be a reliable and unified approach for immunotherapy and relapse prevention in patients with epithelial cancers.

Tolerogenic dendritic cells (tolDCs) and T-regulatory cells (Tregs) are involved in maintaining tolerance to self-antigens and foreign antigens. The cells are used as therapeutic tools for inducing tolerance to transplanted organs or tissues. We investigated the possibility of inducing Tregs in splenocyte cultures using DCs transfected with a DNA construct encoding mouse interleukin-10 (DCpIL-10). DCs were derived from bone marrow cells in the presence of rmGM-CSF and rmIL-4 and electroporated with a plasmid encoding mouse IL-10. Furthermore, DCpIL-10 was cocultured with syngeneic splenocytes. The CD4+CD25hiFoxP3+ Treg frequency, IL-10 expression, and inhibition of the mixed lymphocyte reaction were evaluated. C57Bl/6 and CBA mice differ in their initial frequency of CD4+CD25hiFoxP3+ Tregs and baseline IL-10 production. Also, the effectiveness of CD4+CD25hiFoxP3+ Treg upregulation by tolDCpIL-10 was different. In this study, DCpIL-10 from C57Bl/6 mice induced CD4+CD25hiFoxP3+ Tregs in syngenic splenocytes, which was accompanied by an increase in the IL-10 production and a decrease in the proliferation of splenocytes in response to the alloantigen. DCpIL-10 may be used to induce CD4+CD25hiFoxP3+ Tregs and the regulatory potential of splenocytes.

Background B220+CD11c+plasmacytoid DCs(pDCs) are known to participate in the negative selection and central tolerance induction by the capturing of self-antigens in peripheral tissues and further migration to the thymus using the CCL25-CCR9 chemotaxis axis. Aim Here we investigate the possibility of DCs migration stimulation to the thymus by the transfection with plasmid DNA-constructs encoding CCR9(pmaxCCR9) to develop a system for desired antigen delivery to the thymus for central tolerance induction. Methods Dendritic cells(DCs) cultures were generated from UBC-GFP mice bone marrow cells expressing green fluorescent protein using the rmFlt3-L. DCs cultures were transfected with pmaxCCR9 by electroporation. The efficiency of electroporation was confirmed by RT-qPCR and flow cytometry. The migration of electroporated DCs was assessed in vitro and in vivo. Results Dendritic cells(DCs) cultures obtained from UBC-GFP mice contained both B220+pDCs and SIRPa+cDC2. According to the RT-qPCR assay, the electroporation of obtained DCs cultures with pmaxCCR9 resulted in a 94.4-fold increase of RNA encoding CCR9 compared with non-electroporated cultures. Flow cytometry data showed that DCs cultures electroporated with pmaxCCR9 contained a significantly higher frequency of DCs carrying significantly higher levels of surface CCR9. Migration dynamics of obtained DCs analyzed in vitro showed that pmaxCCR9 electroporated DCs migrated significantly more active to CCL25 and thymic cells than non-electroporated and mock-electroporated DCs. In vivo, 30 days after injection, the relative amount of the DCs electroporated with pmaxCCR9 and pmaxMHC encoding antigenic determinants in the mice thymuses was 2.02-fold higher than the relative amount of the DCs electroporated with control plasmid. Conclusion Thus, the electroporation of murine DCs with pmaxCCR9 stimulated its migration to CCL25 and thymic cells in vitro as well as to the thymus in vivo. The obtained DCs loaded with a desired antigen may be suggested for further evaluation of central tolerance induction ability in in vivo models of autoimmune diseases and transplantation.

Introduction . Currently, new directions in cancer therapy are actively developing, one of which is oncolytic immunotherapy. This approach would be to use of viruses as cancer specific cytolytic agents capable of stimulating both the tumor-specific and non-specific immune response. The objective paper was obtain a recombinant vaccinia virus containing genes encoding immunostimulating molecules and study oncolytic and immunostimulating properties of recombinant virus. Material and methods . MTT test, ELISA, methods of transient dominant selection. Results . The recombinant vaccinia virus (L-IVP_oncoB) were obtained with deletion of the gene encoding thymidine kinase and had an integrated gene encoding GM-CSF. Also the virus have deletion of the gene encoding viral growth factor and integrated genes encoding synthetic tumor-specific polyepitopic immunogens. It was shown that the modifications made to the viral genome did not affect the growth characteristics of the virus when cultured on CV-1 and 4647 cell cultures, and the cytopathogenic efficacy of the virus was determined in relation to cancer cultures of cells of various genesis. In in vivo experiment, it was revealed that the polyepitopic construct in the genome L-IVP_oncoB is able to initiate a change in the profile of cytokines. Discussion . The obtained data characterized L-IVP_oncoB as a promising cytopathogenic and immunostimulating agent and showed the need for further study of its properties as means of oncolytic immunotherapy. Conclusion. The basic experiments on the evaluation of the biological properties of the obtained L-IVP_oncoB, which are necessary for the characterization of the oncolytic virus, have been carried out.

Tolerogenic dendritic cells (tolDCs) and T-regulatory cells (Tregs) are involved in maintaining tolerance to self-antigens and foreign antigens. The cells are used as therapeutic tools for inducing tolerance to transplanted organs or tissues. We investigated the possibility of inducing Tregs in splenocyte cultures using DCs transfected with a DNA construct encoding mouse interleukin-10 (DCpIL-10). DCs were derived from bone marrow cells in the presence of rmGM-CSF and rmIL-4 and electroporated with a plasmid encoding mouse IL-10. Furthermore, DCpIL-10 was cocultured with syngeneic splenocytes. The CD4

The introduction of foreign peptides into alkaline phosphatase that have a minimal influence on the enzymatic activity of a protein is described in the work. Calculated based on the methods of molecular dynamics the longest surface loops of alkaline phosphatase, which are not adjacent to both active center of a dimeric enzyme and contact surface between its monomers, were used as sites of the introduction of foreign peptides. The length of loops was estimated by several methods. Two loops were used to genetically engineer the introduction of peptides. Experiments demonstrated that the introduction of several peptides after the Ala218 residue of alkaline phosphatase insignificantly influences on the enzyme activity. According to experimental data, the selection of the loop for introducing the foreign peptide can be determined using the methods of molecular dynamics based on calculating the mobility of dihedral angles. In order to create macromolecules with new features by introducing foreign polypeptides in enzymatically active proteins, it is possible in practice to use an estimation of the loop length by means of the methods of molecular dynamics.

Advances in oncoimmunology related to the definition of the basic mechanisms of the formation of antitumor immune response, as well as the opening of tumor-associated antigens recognized by immune cells, allowed to start developing ways to influence the effector cells of the immune system to generate effective antitumor cytotoxic response. We investigated the possibility to stimulate an antitumor response in a culture of mononuclear cells of breast cancer patients by dendritic cells transfected with HLA-A*02:01-restricted DNA constructs. We isolated dendritic cells from peripheral blood monocytes and delivered our constructs to these cells by magnetic transfection. Additionally, a series of experiments with loading of dendritic cells with autologous tumor cell lysate antigens was conducted. We have shown that dendritic cells transfected with the HLA-A*02:01-restricted DNA constructs are effective in inducing an antitumor response in a culture of mononuclear cells of breast cancer patients. Dendritic cells transfected with DNA constructor dendritic cells loaded with lysate antigens revealed a comparable stimulated cytotoxic response of mononuclear cells to these two ways of antigen delivery. We conclude that using DNA constructs in conjunction with patient stratification by HLA type allows the application of transfected DCs as an effective method to stimulate antitumor immunity in vitro.

A method for identification of fragments with high local similarity to human proteins within potentially immunopathogenic regions of HIV-1 proteins was developed. The method is based on the use of an original matrix of antigenic similarity of amino acids. The regions whose fragments are frequent in human proteins, and regions exhibiting high similarity to the proteins responsible for important physiological functions, were identified in HIV-1 proteins. A possibility of participation of such regions in immunopathogenesis of HIV infection either through induction of cross-reacting effectors of the immune system or through molecular mimicry of physiologically important human proteins, leading to alteration of homeostasis of the organism, is discussed. Most of the regions identified in HIV-1 proteins contain either T-cell (CD8+ CTL or CD4+ Th) or B-cell epitopes, or both of them simultaneously. The criteria for the design of safe polyepitopic antiviral vaccines that would allow exclusion of epitopes with (immuno)pathogenic potential are discussed. According to these criteria, polyepitopic immunogens should be free of the virus protein regions whose fragments are frequent in human proteins, as well as of regions exhibiting pronounced local similarity to proteins that provide for important physiological functions.

Detailed studies of the pathogenesis of certain neuroviral infections allow for a better understanding of the special role of the olfactory neuroepithelial cells in the invasion of viruses into the CNS. Several studies using animal models demonstrated that neurotropic viruses belonging to various families invade the brain via the olfactory tract after parenteral infection. We suppose that intranasal (i.n.) immunization inducing mucosal and systemic immunity will block neurotropic virus propagation into the brain via the olfactory pathway and neutralize virus multiplication in visceral organs. Subject of the present study was the efficacy of i.n. immunization of Balb/c mice with both killed tick-borne encephalitis (TBE) virus and antigenic peptide 89–119 from the envelope protein E of TBE virus in a nanoemulsion formulation. Intranasal immunization with nanoemulsion containing inactivated TBE virus particles induced specific both neutralizing and HAI antibodies. TBE virus specific IgA antibodies were detected in lung and nasal lavages of mice. The fourth i.n. immunization resulted in a drastic titer increase of the specific antibodies to 1:12,800, which was protective in all i.n. immunized mice against intraperitoneal (i.p.) challenge with 100 LD50 of TBE virus. The ratio of specific IgG2a to IgG1 indicated the occurrence of a Th2 type immune response. We observed a similar balance of TBE virus-specific IgG2a/IgG1 after s.c. immunization of mice with the commercial FSME-Immun Inject® vaccine against TBE virus. Thus, the experimental data obtained for the first time demonstrates the feasibility of using nanoparticles containing inactivated TBE virus for effective protection of i.n. immunized mice against the usually lethal infection. We analyzed a number of fragments of E protein of the TBE virus for antigenic similarity with human proteins by computer analysis. Local antigenic similarity of the fragments of E protein of TBE virus and human proteins allows for the identification of the epitopes, which may induce an autoimmune response if applied in a vaccine construct. One of the candidates, peptide 89–119 of E protein of the TBE virus does not contain any epitopes with local similarities to human protein epitopes. This peptide was synthesized and applied intranasally in nanoparticulate formulation. It induced TBE virus-specific antibodies and led to protection in the case of i.p. challenge with TBE virus.

Background: The safety of a potential AIDS vaccine is an issue that will become critical at later stages of product development and needs to be addressed before it is too late. Objective: In order to design safer vaccine, the HIV antigens, to be deployed in it, should be free of regions that are either present in human proteins or exhibit pronounced structural similarity to proteins responsible for important physiological functions. Study design: The approach is based on the use of an original matrix predicting the antigenic similarity of amino acids. This mathematical approach developed by us was applied for identification of fragments with similarity to human proteins within potentially immunodominant regions of HIV proteins. A potential self-sensitization by viral quasispecies with variants of hypervariable V3 region, generated as a result of immune pressure on the immunodominant region of envelope, was considered in detail. Results: Viral fragments occurring in normal human proteins as well as regions exhibiting high similarity to proteins responsible for physiological homeostasis were identified in every HIV protein at a frequency higher than expected. Most such regions contained either T-cell (CD8+ CTL or CD4+ Helper) or B-cell epitopes, or both of them simultaneously. The gained knowledge was applied in designing a synthetic immunogen containing multiple CTL epitopes. The synthesis of series of chimeric peptides representing hypervariable region of V3 loop of HIV envelope, to be used as a multi-epitope or mixotope vaccine candidate, has been achieved. Such a vaccine could theoretically pre-empt any escape mutant borrowing from antigenic diversity of hypervariable region of V3 loop of HIV envelope. Conclusions: The epitopes shared by HIV and its host are likely to be implicated in the immunopathogenesis of AIDS through induction of cross-reacting effectors of the immune system. The prospect that ‘house-keeping’ immune mechanism can be foiled by molecular mimicry of HIV with physiologically important human proteins should be taken into consideration in safer vaccine design.